Process for preparing variant polynucleotides

ABSTRACT

The present invention discloses a process for the preparation of variant polynucleotides using a combination of mutagenesis of a starting population of polynucleotides and recombination of the mutated polynucleotides. The process comprises the steps of subjecting a population of polynucleotides to (a series of) two (or more) separate PCR&#39;s comprising a first PCR with a forward mutation-specific primer for a position to be mutated and a reverse universal primer and a second PCR with a forward universal primer and a reverse mutation-specific primer for a position to be mutated. The products of the (series of) two (or more) PCR&#39;s are assembled by a polymerase, preferably in one tube.

BACKGROUND OF THE INVENTION CROSS-REFERENCE TO RELATED APPLICATION

This application is the national phase of PCT application PCT/EP02/08225 having an international filing date of 23 Jul. 2002, whichclaims priority from European applications Nr. 01203457.5, filed 11 Sep.2001 and Nr. 01202821.3, filed 23 Jul. 2001. The contents of thesedocuments are incorporated herein by reference.

Protein engineering technology includes the creation of novel proteinsby targeted modificaton(s) of known proteins. However, an approachdirected to targeted modification is only applicable to proteins orprotein families of which the three-dimensional structure of the proteinor at least one member protein of the family has been resolved.Furthermore, many attempts to alter the properties of enzymes by thisapproach have failed because unexpected changes in the structure wereintroduced. If random mutagenesis is applied to create modifiedproteins, it appeared that successfully modified proteins oftenpossessed amino acid substitutions in regions that protein modelingcould not predict.

Various approaches have been developed to mimic and accelerate nature'srecombination strategy to direct the evolution of proteins to morebeneficial molecules. Direct evolution is a general term used formethods for random in vitro or in vivo homologous recombination of poolsof homologous polynucleotides. Several formats are described, forinstance random fragmentation followed by polymerase-assisted reassembly(WO9522625), in vivo recombination (WO97/07205, WO98/31837) or staggeredextension of a population of polynucleotide templates (WO97/07205,WO98/01581). In this way an accumulation of beneficial mutations in onemolecule may be accomplished.

The method of the present invention advantageously enables themutagenesis of a polynucleotide and the random combination of mutatedpositions to be performed in one process, without the necessity forprior fragmentation of the polynucleotide. The method is reproducible,highly controllable and very fast. A further advantage of the method ofthe invention is that the recombination frequency is high and the chanceto re-isolate the starting polynucleotide is low.

DETAILED DESCRIPTION

The present invention provides a method for the preparation of a variantpolynucleotide.

The method according to the invention comprises the steps of:

-   subjecting a population of polynucleotides to two or more separate    PCR's, a first PCR with a forward mutation-specific primer directed    to a position to be mutated and a reverse universal primer, a second    PCR with a forward universal primer and a reverse mutation-specific    primer directed to a position to be mutated, and, optionally, a    third or-   more PCR with a suitable forward and reverse primer;-   assembling the products of the two or more PCR's by a polymerase;-   optionally amplifying the assembled polynucleotides;-   preparing a library of the resulting variant polynucleotides;-   screening said library of variant polynucleotides for a variant    polynucleotide with a desired property.

A variant polynucleotide is defined herein as a polynucleotide differingin at least one position from any one of the members of the populationof polynucleotides that forms the starting material for the processaccording to the invention.

The population of polynucleotides that forms the starting material forthe process according to the invention comprises polynucleotide membersthat display a substantial homology to each other. A substantialhomology is defined herein as a homology from 70-100%, preferably from75-100%, preferably from 80-100%, preferably from 85-100%, morepreferably from 90-100%, most preferably from 95-100%. A population ofpolynucleotides comprising polynucleotide members displaying asubstantial homology for instance may be a population of polynucleotideswherein the polynucleotide members are identical polynucleotides, and/orare mutants of a parental polynucleotide and/or are members of a genefamily.

A population of identical polynucleotides may for instance be apopulation of polynucleotides encoding a wild type polypeptide.

A population of mutants derived from a parental polynucleotide maycomprise different mutants, each individual mutant in the populationdiffering in at least one position from the parental polynucleotide. Apopulation of different mutants derived from a parental polynucleotidemay be obtained by methods known in the art. For instance, the mutantsmay be obtained by classical random or site-directed mutagenesistechniques. A suitable random mutagenesis technique for Instance is theerror-prone PCR technique.

The population of mutants may comprise mutants that have been previouslyscreened and selected for a certain desired property.

A population of members of a gene family typically contains differentmembers of a gene family, i.e. polynucleotides displaying a considerablesequence homology, i.e. at least 70%, and having a similar function inan organism. For instance, such polynucleotides may encode relatedproteins originating from different strains, different species,different genera, different families. An example is the phytase genefamily from the genus Aspergillus, displaying a homology of at least 90%within the species Aspergillus niger.

The starting population of polynucleotides may conveniently be subjectedto the process of the invention when being cloned in a vector and/or asisolated fragments. In a situation that the starting population ofpolynucleotides is obtained by a prior screening and selection process,the vector may conveniently be an expression vector.

According to the method of the invention, the starting population ofpolynucleotides is subjected to two or more separate polymerase chainreactions (PCR's), a first PCR with a forward mutation-specific primerfor a position to be mutated and a reverse universal primer, a secondPCR with a forward universal primer and a reverse mutation-specificprimer for a position to be mutated, and, optionally, a third or morePCR with a suitable forward and reverse primer.

The first PCR generates fragments with a common 3′ end and different 5′ends, the 5′ end being determined by the position of the forwardmutation-specific primer within the starting polynucleotide sequence.The second PCR generates fragments with a common 5′ end and different 3′ends, the 3′ end being determined by the position of the reversemutation-specific primer within the starting polynucleotide sequence.

The third or more PCR may generate fragments that, for instance, arepositioned in between the fragments obtained in the first and secondPCR. A third or more PCR may be applied, for instance, if the startingpopulation of polynucleotides comprises members which size is such thatthe application of two PCR's will result in an inconveniently low yieldof full-length assembled product after the assembly (fusion) PCR.Suitable forward and reverse primers for the third or more PCR arechosen such that the resulting PCR product(s) will overlap with theadjacent 5′ and 3′ fragments. The primers suitable for the third or morePCR may contain a mutated position.

Preferably, any forward mutation-specific primer has a correspondingreverse mutation-specific primer, i.e. sets of mutation-specific forwardand reverse primers are designed directed to the same nucleotideposition(s) within a polynucleotide member of the starting population ofpolynucleotides.

Preferably, a series of two or more separate PCR's is performed usingone set of forward and corresponding reverse mutation-specific primerper separate PCR, resulting in a series of separate PCR products.

The mutation-specific primer is designed in such a way that at least onenucleotde position within the primer differs from the correspondingnucleotide position within a member of the starting population ofpolynucleotides. This (these) position(s) is (are) called the mutatedposition(s). Typically, the mutated position(s) within the primer is(are) located at least one nucleotide from the end of the primer. Thenumber of mutated positions per primer and the length of the primer arenot critical to the invention and may conveniently be determined on acase by case basis. The number of mutated positions per primer forinstance may depend on the length of the primer, whereas the length ofthe primer may depend on the type of primer used (e.g. saturated orspiked).

In one embodiment of the invention, a (series on mutation-specificprimer(s) is designed in such a way that a mutated position in a primercorresponds to a selected position, e.g. a position that is shown to bean effective mutation, in a member of the starting population ofpolynucleotides.

In another embodiment of the invention, a (series of) mutation-specificprimer(s) is designed in such a way that the primer(s) ensure theintroduction of mutations at random positions within the startingpopulation of polynucleotides.

In still another embodiment of the invention, the mutation-specificprimer is a saturated primer, i.e. a primer designed to introducealtered nucleotides in a triplet encoding an amino acid such that morethan one amino acid substitution is introduced at a particular positionin the polypeptide encoded by the polynucleotide to be modified.Preferably, the saturated primer is designed such that a substitution ispossible of an amino acid of choice with any of the other 19 amino acidsnaturally occurring in a protein. A saturated primer typically has alength of about 18-30 nucleotides. A saturated primer effectivelycomprises a mixture of individual oligonucleotides wherein anyindividual oligonucleotide enables substitution of a chosen amino acidfor any one of the other amino acids.

In still another embodiment of the invention, the mutation specificprimer is a spiked primer (or spiked oligonucleotide). A spikedoligonucleotide contains, per nucleotide to be mutated, for instance anucleotide within a codon, a majority of the original nucleotide and aminority of the nucleotide to be substituted. A suitable percentage forthe majority of the original nucleotide is 80-99%. The length of aspiked oligonucleotide may depend on the length of the polynucleotide tobe mutated and/or the number of desired mutations. It is convenient thatat the ends of a spiked oligonucleotide, at least two nucleotides willbe 100% similar to the original nucleotide sequence, i.e. will not bemutated.

For instance, an oligonucleotide of 55 nucleotides containing 5nucleotides at both ends that are not mutated and being spiked with apercentage of 4.5% over the remaining 45 nucleotides will yield abouttwo substitutions per oligonucleotide.

Of course, the skilled person is very well able to combine any of theembodiments mentioned above if desired.

The universal primers used in the process of the invention may bedirected to the vector and/or to the polynucleotide to be used.Preferably, the universal primers contain a unique restriction enzymesite enabling the cloning of the resulting variant polynucleotides in asuitable vector.

According to the invention, the products of the two or more separatePCR's are mixed, preferably in equimolar amounts, and assembled by apolymerase, in a so-called assembly or fusion PCR. Optionally,additional universal forward and reverse primer may be added to theassembly reaction mixture to simultaneously amplify the assembledpolynucleotides by PCR.

In one embodiment of the invention, the series of separate PCR productsobtained from a series of two or more separate PCR's are assembled by apolymerase in one tube, in other words subjected to a one-tube fusionPCR. Preferably, the separate PCR products are mixed in equimolaramounts.

In another embodiment of the invention, the series of separate PCRproducts obtained from a series of two or more separate PCR's areseparately assembled by a polymerase, in other words subjected toseparate fusion PCR's. A separate fusion PCR's is performed with theproducts of the first, second and optionally third PCR. This embodimentmay be advantageous if a set of forward and reverse mutation-specificprimer contains two or more mutations per primer.

In a situation that mutations are desired only near the end(s) of apolynucleotide, it may be an option to apply one PCR that effectivelycombines the two or more separate PCR's and the fusion PCR in onereaction.

The method of the invention advantageously enables the mutagenesis of apolynucleotide and the random combination of mutated positions in oneprocess. In addition, the present invention advantageously enables theoption to isolate “intermediate” variant polynucleotide products bysubjecting the products of any desired set of forward and reversemutation-specific primer to separate fusion PCR's. (Selected) variantpolynucleotide resulting from several separate PCR's according to theinvention can then be used as starting material for a further PCRaccording to the invention.

Any of the PCR's as performed in the method of the invention may beperformed following conditions generally known to the person skilled inthe art. The conditions typically may depend on the primers and theenzyme used. It may further be an option to perform any of said PCR'sunder error-prone conditions, i.e. under conditions that reduce thefidelity of nucleotide incorporation, thus randomly introducingadditional mutations in the variant polynucleotides obtained by themethod of the invention. Error-prone conditions may for instance beprovided by independently varying the concentrations of manganese anddGTP in the PCR reaction. Typically, the mutagenesis rate may be raisedby increasing the amount of manganese and/or dGTP in the PCR reaction.

The polynucleotide products of the assembly reaction are cloned in asuitable vector, to enable the preparation of a library of variantpolynucleotides. The choice of the vector will depend on the hostwherein the library is propagated. Subsequently, the library of variantpolynucleotides is screened with a suitable screening method to enablethe selection of a variant polynucleotide with a desired property.

The method used for screening the library of variant polynucleotides isnot critical for the invention. Typically, the method used will dependon a property of the polynucleotide of interest. If the polynucleotideof interest comprises a gene encoding a polypeptide, a suitablescreening method may be directed to directly assay said polypeptide. Asuitable screening method may further be directed to assay a primary orsecondary metabolite if the polypeptide is an enzyme involved in theproduction of said primary or secondary metabolite, for instance anenzyme that is part of the biosynthetic pathway of said metabolite.Examples of such metabolites are an amino acid, a vitamin, anantibiotic, a carotenoid.

The method of the invention is suitable for the mutagenesis of anypolynucleobde of interest.

In one embodiment of the invention, the polynucleotde of interestcomprises a gene encoding a polypeptide. Said polypeptide may forinstance be a structural protein, a peptide hormone, a growth factor, anantibody or an enzyme. The polypeptide may be produced intracellularlyor may be secreted from the cell into the environment, for instance theculture medium. The polynucleotide may comprise a single gene or maycomprise a cluster of genes. Said cluster of genes may comprise genesencoding enzymes involved in the biosynthesis of a particular metaboliteand/or genes encoding regulatory factors involved in the regulation ofexpression of one or more genes involved in production of a particularmetabolite.

In another embodiment of the invention, the polynucleofide of interestmay be a non-coding polynucleotide, for instance a regulatory regioninvolved in the control of gene expression, on transcriptional and/ortranslational level. The process of the invention may also be applied toa polynucleobde comprising a gene (cluster) and corresponding regulatoryregions.

The present invention further envisages production of a variantpolypeptde by expressing a variant polynucleotide produced and selectedaccording to the invention in a suitable host organism and, optionally,recovery of the produced polypeptide.

To this end, the selected polynucleotide is cloned in an expressionvector of choice and transformed to a host organism of choice.Transformed host cells are selected from the untransformed background byany suitable means. The transformed cells are grown in a suitableculture medium and may further be screened for expression of the variantpolynucleotide. Techniques for the transformation of host cells and forthe selection of transformed cells are commonly known to the skilledperson.

For production of the variant polypeptide on a larger scale, atransformed cell producing a suitable amount of the variant polypeptideof interest may be cultured under conditions conducive to the productionof said polypeptide. Optionally, the polypeptide may be recovered fromthe culture medium and/or form the host organism. Depending on itsfurther use, recovery of the variant polypeptide may include itsformulation in a suitable liquid or solid formulation, and/or itsimmobilization.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustration of (saturated) Mutation Primer PCR DNARecombination. * mutated position; {circle around (x)} saturatedmutation site

FIG. 2. Agarose gel electrophoresis of the single step PCR's.

Above 1-9: DNA product after a PCR reaction with the universal 5′ primerand mutation primers 1-9.

Under 1′-9′: DNA product after a PCR reaction with the universal 3′primer and mutation primers 1′-9′.

FIG. 3. Typical results of the conversion activities of a group ofmutants from part of the KKN07 library selected after the first MTPanalysis.

EXPERIMENTAL

MTP Screening

Single colonies of the library to be screened were inoculated inindividual wells of microtiter plates (MTP's) filled with SE liquidmedium (containing bacto tryptone 10 g/l, bacto yeast 5 g/l and NaCl 5g/l), supplemented with ampicillin at a final concentration of 100μg/ml. If required, arabinose inducer was added (final concentration0.002%). Normal growth conditions were at 37° C.; induced growthconditions were at 28° C. and 280 rpm. 50 μl of the 20-24 hour growncultures were incubated with D,L-α-methylphenylglycine amide (Femam) at55° C. in deepwell plates. After 2.5 hours off incubation the amidaseactivity of the culture broth was measured by measuring the amount offormed L-α-methylglycine (Femac).

CFE Screening

Cell-free extracts (CFE's) were prepared using a bacterial proteinextraction reagent according to the manufacturers instructions (BPER,Pierce, Rockford, Ill. USA) and their L-amidase activity was measured.

Amidase Activity Assay

Amidase activity was measured as conversion activity from Femam toFemac. Detection occurred by NMR.

Example 1

Preparation and Screening of an Error-prone Library of O. anthropiL-amidase

An error-prone PCR was performed on the Ochrobactrum anthropi L-amidasegene (see SEQ ID No 1) using the Diversify™ PCR Random Mutagenesis kitfrom Clontech (Palo Alto, Calif. USA) according to the manufacturer'sinstructions. The PCR products were cloned In the Eagl/HindIII sites ofthe vector pBAD/Myc-HisC (Invitrogen Corporation, Carlsbad, Calif. USA)and transformed to E. coli Top10F cells (Invitrogen Corporation,Carlsbad, Calif. USA). Clones were first screened on MTP and CFE's of asubset of clones were further screened (see Experimental). Improvedmutants were sequenced to determine the modified position(s). Themodified positions of seven improved mutants are indicated hereinafterV52A, F93V, T143A, T193P, N212D, N981/L124P, K13BR/G234V (see SEQ ID No2).

Example 2

Mutagenesis and Recombination of Improved Mutants by Mutation Primer PCR

Saturation mutagenesis at sites discovered after screening error-pronemutagenesis libraries will introduce all 20 amino acids into thesepositions. Saturated forward and reverse primers were designed for eachmutation of the seven improved mutants obtained in Example 1. Thesaturated primers are designed with three N's at the codon of the aminoacid to be mutated

As can be seen in FIG. 1 two separate PCR reactions for each mutationsite were performed 1) using a forward saturated primer in combinationwith a universal reverse primer, and 2) the reverse saturated primer incombination with a universal forward primer.

The PCR products of these separate PCR reactions (see FIG. 2) were mixedin equimolar amounts and assembled by polymerase. The products of thisso-called fusion PCR reaction were subsequently amplified in anerror-prone PCR. The DNA products were cloned in the Eagl/HindIII sitesof the pBAD/Myc-HisC vector and transformed to E. coli Top10F cells.

5000 clones of the resulting KKN07 library were screened in MTP.Approximately 40% of all the mutants analyzed proved to be inactive inMTP.

A large group of mutants selected from the MTP screening was tested in asecondary screening. In this screening, CVE's and different dilutionsthereof were analysed for conversion activity by NMR. The conversion/μlwas calculated and corrected for the amount of protein. Subsequently theconversion/μl/mg protein of the mutants was compared withconversion/μl/mg protein of the wild type, and the activity improvementwas determined.

In FIG. 3 the overall results of selected mutants from the KKN07 libraryare presented. DNA of 10 randomly picked mutants and the 10 mostimproved mutants was sequenced.

The results of these sequence analyses are presented in Table 1.

From the sequence results of the randomly picked mutants a recombinationfrequency of about 70% was calculated (3 triple, 4 double and 3 singlemutations). The mutation rate of the used error-prone conditions wasapproximately 0.9 mutations/gene.

Using one cycle of mutagenesis by error-prone PCR, saturationmutagenesis combined with recombination and screening/selection of afraction of the library, the specific activity of Ochrobactrum anthropiL-amidase towards Femam was improved by approximately 5-6 times ascompared to wild type

TABLE 1 Sequence results of 10 randomly picked mutants (KKN07-1/10) aswell as 10 of the most improved mutants from the KKN07 library. At thetop of the table the 7 selected mutants that were used as startingmaterial are indicated. The conversion activity is indicated relative towild type. Mutations EP Mutations Conversion activity WT 1   T1 T143A1.8-2.3 T8 F93V 1.5 F10 N98I L124P 1.4 F162 K138R G234V 1.3 F190 N212D1.1 R9 T193P 1.4 R10 V52A 2.0-2.5 KKN07-1 V52A K138R N212C T123R, T273AKKN07-2 F93T R132C, Deletion KKN07-3 V52W N98E T143A KKN07-4 L124T T193PKKN07-5 V52A T143K N212D KKN07-6 V52T Deletion KKN07-7 T143A C285YKKN07-8 T143K N212D KKN07-9 K138* T143L D222N KKN07-10 V52A G234V F306S,A281T F308 V52A T143A T193P G234C R229W 5.6 F317 N98I L124G T193P 4.5F303 V52A T143E 4.5 F386 N98L K138R T193P R132C 4.3 F409 V52A T143AG234Q L240F 4.1 F382 V52A K138R T193P P224T 4   F384 F93V T193P A116T3.7 F389 V52S T193P 3.6 F302 V52S N98V T143A 3.7 F401 T193P N212T 3.6*stands for stop codon

1. A process for preparing a population of variant polynucleotidescomprising at least one polynucleotide having a desired property,comprising: (a) subjecting a starting population of polynucleotidescomprising at least two different mutants of a parental polynucleotideto separate PCR's to result in products of the PCR's, wherein the atleast two different mutants were selected for the desired property: (i)a first set of at least two PCR's, which are performed separately fromeach other, with each PCR using a forward mutation-specific primer whichhybridizes to a mutated position in a mutant of the starting populationand a reverse universal primer to generate a first common end for thefirst set of PCR's, (ii) a second set of at least two PCR's, which areperformed separately from each other, with each PCR using a forwarduniversal primer to generate a second common end for the second set ofPCR's and a reverse mutation-specific primer which hybridizes to amutated position in a mutant of the starting population, and (iii)optionally a third or more PCR with suitable forward and reverseprimers; (b) assembling the resulting products of the PCR's using apolymerase to obtain said population of variant polynucleotides, whereinthe variant polynucleotides differ in at least one position from theparental polynucleotide; and (c) optionally amplifying the population ofvariant polynucleotides.
 2. The process of claim 1, wherein the startingpopulation of polynucleotides displays homology within said populationof 70-100%.
 3. The process of claim 1, wherein at least onemutation-specific primer is a saturated primer.
 4. The process of claim1, wherein at least one mutation-specific primer is a spikedoligonucleotide.
 5. The process of claim 1, wherein the PCR's and/or theamplification of assembled polynucleotides are performed undererror-prone conditions.
 6. The process of claim 1, wherein the at leastone variant polynucleotide having a desired property comprises one ormore gene(s) encoding a polypeptide.
 7. The process of claim 1, whereinthe at least one variant polynucleotide encodes an enzyme involved inbiosynthesis of a primary or secondary metabolite.
 8. A process forproducing a variant polypeptide comprising expressing the at least onevariant polynucleotide prepared according to the process of claim 1 in asuitable host and, optionally, recovering the variant polypeptide.
 9. Aprocess for producing a primary or secondary metabolite comprisingexpressing the at least one variant polynucleotide prepared according tothe process of claim 7 in a suitable host and, optionally, recoveringthe primary or secondary metabolite.
 10. The process of claim 1 whichfurther comprises: (d) preparing a library of said variantpolynucleotides and (e) screening the library for said at least onevariant polynucleotide with a desired property.
 11. The process of claim1, wherein the at least one polynucleotide encodes an enzyme.
 12. Aprocess for preparing a population of variant polynucleotides comprisingat least one polynucleotide having a desired property, comprising: (a)subjecting a parental polynucleotide to mutagenesis to provide mutants;(b) screening the mutants to provide at least two different mutantsselected for the desired property as a starting population ofpolynucleotides; (c) subjecting the starting population ofpolynucleotides comprising the at least two mutants to separate PCR's toresult in products of the PCR's: (i) a first set of at least two PCR's,which are performed separately from each other, with each PCR using aforward mutation-specific primer which hybridizes to a mutated positionin a mutant of the starting population and a reverse universal primer togenerates a first common end for the second set of PCR's, (ii) a secondset of at least two PCR's, which are performed separately from eachother, with each PCR using a forward universal primer to generate asecond common end for the second set PCR's and a reversemutation-specific primer which hybridizes to a mutated position in amutant of the starting population, and (iii) optionally a third or morePCR with suitable forward and reverse primers; (d) assembling productsof the PCR's using a polymerase to obtain said population of variantpolynucleotides, wherein the variant polynucleotides differ in at leastone position from the parental polynucleotide; and (e) optionallyamplifying the population of variant polynucleotides.
 13. The process ofclaim 12, wherein the at least one polynucleotide encodes an enzyme.